Acquired resistance to antibacterial drugs is a growing public health problem due to the spread of multidrug-resistant bacteria. The long-term objective of this project is to build the knowledge to engineer new combinations of polyketide synthase (PKS) catalytic domains with reliable predictions of the chemical and stereochemical outcome. Modular PKSs are among the most desirable target pathways for "combinatorial biosynthesis" because of the rich chemical diversity of their products. They are also among the most amenable pathways to this approach because of their modular organization. Recombination of PKS domains to build synthetic pathways for novel compounds requires an understanding of the basis for substrate specificity and stereochemical outcome for each catalytic domain, which at present is lacking. Crystal structures of some domains are available, but, apart from the preliminary studies for this application, the structures lack bound substrates or analogs. Affinity labels have been demonstrated to be an effective means to establish the mechanism of pikromycin thioesterase (Pik TE). The central hypothesis of the proposed research is that polyketide-based affinity labels are powerful tools to probe the structure and mechanism of isolated PKS didomains. The specific aims of this application are: (1) to determine the structural basis for substrate specificity and stereochemical outcome of several NADP-dependent ketoreductase (KR) domains, (2) to explore the substrate range of KR domains for non-natural substrates, and (3) to understand the mechanisms of macrolactone formation by terminal thioesterase (TE) domains. The research design and methods will utilize the acyl carrier protein (ACP) of didomain constructs, KR-ACP and ACP-TE, to deliver substrate and product mimics to the KR and TE active sites. Natural KR-ACP and ACP-TE didomains will be covalently modified by vinyl ketone affinity label mimics, and their crystal structures determined. Didomains from the PKS systems for pikromycin, erythromycin, and tylosin will be examined since these molecules are established lead compounds for antibiotic drug discovery. The proposed research is significant because the rational engineering of PKS systems is expected to provide novel macrolide antibiotics to combat the rising tide of multidrug-resistant bacteria. PUBLIC HEALTH RELEVANCE: Complex mega-enzyme machines known as polyketide synthases (PKS) produce many antibiotics and other bio-active molecules. With appropriate engineering, these proteins are potentially rich sources of new pharmaceuticals. This project will examine the details of substrate interactions with several PKS domains. Substrate mimics will be synthesized that covalently attach to the enzyme, and crystal structures of substrate mimic trapped in the enzyme will be solved. The goal is to understand how the substrate specificity and range for each reaction, and thereby allow design of new enzymes to make new bio-active molecules.